Thromboxane B2 metabolite methods for optimizing aspirin dosage

ABSTRACT

Disclosed are unique methods for identifying the lowest, yet optimal, aspirin doses for patients. These methods are also characterized as having little to no aspirin-related side-effects. These methods may be used pre- as well as post-thrombotic event, and employs a patient&#39;s urinary thromboxane B 2  metabolic levels (e.g., 11-dehydrothromboxane B 2 ), to identify the patient&#39;s platelet activation level. A patient&#39;s urinary thromboxane B 2  metabolic level is then used to calculate and appropriate and individualized treatment effective for utilizing platelet activation. Kits for utilizing this technique are also provided. In yet another particular aspect, the invention provides a method for utilizing a random urine sample obtained from a patient to determine whether a patient or particular individual&#39;s current dosage of aspirin is providing an adequate and appropriate level of inhibition of platelet activation levels, as compared to inhibition levels observed in individuals not taking aspirin.

This application is a continuation of application Ser. No. 09/619,211,filed Jul. 19, 2000, now abandoned, which claims the benefit of U.S.Provisional patent application 60/161,462, filed Oct. 25, 1999.

BACKGROUND OF THE INVENTION

Aspirin (acetyl salicylic acid) effectively reduces the risk ofsecondary thrombotic events in individuals who have experienced angina,myocardial infarction, peripheral artery disease, or cerebrovascularischemia. Aspirin also may reduce the risk of initial thrombotic eventsin healthy individuals. For this reason, many individuals, throughphysician prescriptions or self-medication, take aspirin on a regularbasis for the primary or secondary prevention of thrombotic disease.

Some important questions remain unanswered. What long-term dosage ofaspirin is likely to confer protection from thrombosis while avoidinggastric discomfort or hemorrhagic conditions? What individuals are“resistant” to aspirin, how may aspirin resistance be identified, andwhat effect does aspirin resistance have on the interpretation ofclinical trials?

Several studies suggest that aspirin, at doses between 30 and 325mg/day, is effective in reducing the incidence of arterial thromboticevents. Physicians customarily prescribe aspirin to prevent myocardialinfarction, cerebrovascular thrombotic disease, and vascular death inindividuals with stable angina (1), unstable angina (2), myocardialinfarction (3), transient cerebral ischemia, peripheral vasculardisease, and thrombotic stroke. (4) For patients with prior acutemyocardial infarction or stroke, aspirin prevents up to 40 newthrombotic events per thousand treated. (5) Two large randomized trialsdemonstrate the thrombosis-reducing efficacy of aspirin for patientswith acute ischemic stroke, and aspirin is now routinely prescribed inthese conditions. (6,7)

Aspirin has been shown to be effective in prevention of heart disease inmen with several high risk clinical conditions and both men and womenwith hypertension. (8,9) The Program on the Surgical Control of theHyperlipidemias categorized a number of individuals who had a partialileal bypass surgery as smokers and non-smokers and assigned them toaspirin and non-aspirin arms. Within this population, the overallmortality rate was 45.2% for smokers with no aspirin use and only 10.4%for those who reported even infrequent aspirin use. (10)

Primary Prophylaxis with Aspirin

Aspirin reduces the incidence of thromboembolic arterial disease inhealthy individuals over 50 years of age. (11, 12, 13) In healthy men,aspirin appears to prevent an average of four thrombotic events perthousand subjects treated. In the Physicians' Health Study, healthy menwho took 325 mg of aspirin every other day experienced a mean reductionin the incidence of first myocardial infarction of 44.8% compared tothose taking placebos. (14) There was a particularly marked reduction of59.3% for the morning peak of infarction, between 4 and 10 a.m. Thesefindings were not confirmed in the British Doctors' study, however, andadditional confirmatory studies are in progress, including the currentWomen's Health Study. (15)

Aspirin Dosage and Complications

Hemorrhage and Gastrointestinal Toxicity

There is a dose-related risk of gastrointestinal bleeding in aspirintherapy, especially in patients who have coagulopathy or who are takingadditional anticoagulant therapy such as heparin or warfarin. (16) Thisis of particular concern in individuals with stomach lesions such as theulcers associated with Helicobacter pylori infection. To avoidhemorrhage, many physicians recommend enteric-coated aspirin, especiallyif the recommended dosage exceeds 325 mg/day. (17) In the BritishDoctors' Study, where the prescribed randomized dose was 500 mg/day, 20%of aspirin arm participants dropped out due to dyspepsia orconstipation, 3.6% experienced bleeding or bruising, and 2.2% hadgastrointestinal blood loss. (18)

In the Cardiovascular Health Study, Kronmal, et al found a 1.6× relativerisk of ischemic stroke and a 4× relative risk for hemorrhagic strokefor healthy women 65 and older who took aspirin. (19) This studypresents self-reported, not randomly assigned, aspirin users andillustrates the hemorrhagic risk of high-dose aspirin in particulardisease situations.

For patients who have an elevated risk of thrombosis, the absolutebenefit of aspirin prophylaxis clearly outweighs the relatively smallrisk of bleeding. However, the individuals with no risk factors, aspirindosages must be carefully monitored to avoid gastric discomfort, gastrichemorrhage, systemic hemorrhage and hemorrhagic stroke.

Recommendations for Therapeutic Aspirin Usage

Because of the preponderance of evidence favoring the protective effectsof aspirin against arterial thrombosis, millions rely on aspirinprophylaxis daily either by physician's prescription or self-medication.In a review of clinical studies undertaken in the 1980s and 1990s,Hirsh, Dalen, Fuster, et al make the following recommendations: (4)

-   -   Aspirin is indicated for patients with stable angina, unstable        angina, acute myocaridal infarction, transient cerebral        ischemia, thrombotic stroke, and peripheral arterial disease.    -   A dose of 75 to 100 mg/day should be used chronically for all        indications, although an initial dose of 160 to 325 mg should be        used in acute settings.    -   For patients with cerebrovascular disease, a dose of 75 mg/day        is effective.    -   Aspirin at 100 mg/day is indicated for patients with prosthetic        heart valves who develop systemic embolism while on warfarin.    -   Aspirin is indicated for patients with atrial fibrillation in        whom warfarin is contraindicated.        There is no recommendation either for or against aspirin usage        in normal, healthy adults, and no standards have been        established for laboratory monitoring of aspirin's efficacy, a        growing concern as clinicians became aware of aspirin resistance        and interindividual pharmacokinetic variations.

Recent clinical investigations indicate that approximately 10% to 15% ofpatients on aspirin therapy for prevention of thrombosis have a lessthan adequate platelet suppression response. (20) Additionally, it isreported that some individuals develop an increasing resistance overtime. (21) It is uncertain if this observed resistance is the result ofpoor absorption, changes in pharmacodynamics, non-compliance, ormechanisms not currently identified. Ridker, et. al. reported that thepositive effect of aspirin appears to be altered by underlyinginflammation and further suggested that markers of inflammation such asC-reactive protein may delineate those individuals who will respondatypically to aspirin. (22)

Platelet Activity Studies and Variable Aspirin Response

Current laboratory measures of platelet activation include the Mielketemplate bleeding time, aggregometry, lumiaggregometry, the Dade-BehringPFA-100 platelet function analyzer, and the platelet reactivity test.(23) While the platelet-suppressing property of aspirin clearly affectsthe results of these procedures in general, individuals' laboratoryresponses to aspirin therapy are idiosyncratic. Trip et. al. report 46%of patients with positive spontaneous platelet aggregation resultssuffered a repeat myocardial infarction. (24) This was the first studythat paired clinical outcomes with laboratory results. Helgason, et. al.demonstrated that seven of 17 patients with atrial fibrillation achievedonly partial inhibition of platelet aggregation when taking 325 mg ofenteric coated aspirin per day. Non-compliance had no statistical effecton this study's outcome. Helgason's group further demonstrated that 8.2%of patients with previous ischemic stroke exhibited restoration ofplatelet aggregation (aspirin resistance) despite escalation of aspirindosage to, ultimately, 1300 mg/day. (25)

Pappas, et al used a specially designed visual platelet aggregateinspection technique to measure the response to the inhibitory effectsof aspirin in 31 healthy young adults. (26) They demonstrated a wideinter-individual variation in response to aspirin that was consistentover 28 days of aspirin ingestion. Mueller, et al performed correctedwhole blood aggregometry on patients with intermittent claudication whowere taking 100 mg aspirin/day after elective peripheral balloonangioplasty. (27) At any given time, only 40% of males showed completeinhibition of aggregation. Significantly, non-responsive aggregometryresults in this study predicted increased risk of reocclusion, leadingto the conclusion that aspirin may fail to protect partial-andnon-responders from occlusive events.

Grotemeyer, et al, performed whole platelet reactivity tests on 180internal carotid artery stroke victims given 500 mg of aspirin threetimes per day. (28) All began with elevated platelet reactivityimmediately following stroke. Upon initial aspirin administration, 90%of the subjects demonstrated an immediate suppression of plateletreactivity. However, 60 patients' platelets resumed enhanced plateletreactivity only twelve hours after the initial aspirin dosage. Thesewere termed secondary aspirin non-responders. Over a 24-month periodfollowing discharge, 24 (40%) of the secondary aspirin non-respondersexperienced myocardial infarction, repeat stroke, or vascular death(p<0.0001). Of 114 remaining subjects (six were lost to follow-up), onlyfive (4.4%) suffered these major endpoints. Grotemeyer concluded thatearly identification of secondary aspirin non-responders is an importantstep to effective prevention of further thrombotic events in post-strokepatients. In an extensive review of aspirin and platelet laboratorystudies, Patrono, et al, commented that 10% to 15% of individuals have apoor initial response or demonstrate progressive resistance to aspirin.No large clinical trials have incorporated laboratory measures ofplatelet activation, so the effect of aspirin resistance on clinicaloutcomes is currently unknown.

Komiya, et al used platelet aggregometry to detect cases of aspirintherapy non-compliance and incorrect dosage. They found that 10% of 159outpatients' results were outside the diagnostic parameters because ofnon-compliance and that an additional 2% were confirmed compliant butstill had normal aggregometry results. (29) Their study illustrates thenecessity for monitoring aspirin therapy in patients who may besuspected of non-compliance.

Aspirin Suppresses Platelet Activity

Agonists Trigger Platelet Activation

The cyclooxygenase (COX) biochemical activation pathway, as diagrammedin FIG. 1, is essential to normal platelet activation and to theprevention of systemic hemorrhage. (30) COX is also an importantactivation enzyme in other cells.

Activation begins when a platelet agonist such as ADP, epinephrine,collagen, or thrombin binds to its platelet membrane receptor site. Thisactivates phospholipase A₂, a membrane-associated enzyme, and freesarachidonic acid, a 20-carbon unsaturated fatty acid, from itssupporting membranes phospholipid. Free arachidonic acid is a substratefor the COX pathway. (31)

The Platelet Cyclooxygenase Pathway

COX, a membrane-associated endoperoxide synthase with two catalyticsites, rapidly modifies the free arachidonic acid in a two-step process.(32) The first catalytic site converts it to the endoperoxide PGG₂. Thesecond site, a peroxidase-type site, converts the short-lived PGG₂ toPGH₂. PGH₂ is then converted by the isomerase action of thromboxanesynthase to thromboxane A₂ (TXA₂), which activates the platelet.

TXA₂ is rapidly hydrolyzed to thromboxane B₂ (TXB₂), a stable plasmaproduct of the COX pathway. TXB₂, in turn, is converted to a variety ofend products, most of which are excreted via the kidney. (33)

Aspirin Irreversibly Acetylates Cyclooxygenase

Platelets (and other cells) are now known to produce two isoforms ofCOX-1 and COX-2. (34) COX-1 is a constitutive membrane-bound enzyme thatfunctions in all normal platelets, whereas COX-2 is a cytokine-inducibleenzyme that appears in newly produced platelets and in other cellsduring inflammation.

Aspirin irreversibly acetylates both COX-1 and COX-2 at serine 529, seeFIG. 2. For the COX-1 enzyme, the attached acetyl group stericallyhinders arachidonic acid's access to its reactive site. Acetylation doesnot appear to hinder the activity of COX-2. The inflammation-inducedactivity of COX-2 in platelets may account for some cases of aspirinresistance (35), as may pharmacokinetic variations among individuals.

Aspirin Pharmacokinetics

Aspirin is rapidly absorbed from the stomach and duodenum and is rapidlyhydroyzed to salicylic acid by esterases in the gut, liver, anderythrocytes. Because only aspirin, not salicylic acid, acetylates COX-1(or COX-2), a significant proportion of acetylation occurs in thepresystemic circulation of the gut and liver. (36) Salicylic acidcirculates bound to plasma proteins for up to six hours and is clearedby the kidney.

Platelets acetylated during the time of peak aspirin levels lose most oftheir ability to be activated; as reflected in prolonged bleeding times,reduced aggregometry responses, and diminished TXB₂ production. A singledose of 325 mg aspirin is detectable within minutes using theselaboratory assays and the effects remain for six to ten days. Plateletswith acetylated COX-1 survive normally and continue to participate inthe adhesion reaction. Normal platelet function test results arerestored only when a predominant population of new platelets has beenreleased from the bone marrow.

Additional Platelet Activity-Suppressing Substances

Other non-steroidal anti-inflammatory drugs (NSAIDs) such asdipyridamole, sulphinpyrazone, and ibuprofen act upon COX-1 or otherplatelet enzymes, but no clinical trials have established antithromboticproperties for these drugs. Ticlopidine, clopidegril, and abiximab exerttheir antiplatelet activity on membrane receptors. The effects of allthese therapeutics may be measured using aggregometry, lumiaggregometry,and plasma, serum, or urine TXB₂ assays.

In addition, many dietary components and supplements have been shown tomodify platelet function by unknown mechanisms. These include fish oil(37), vitamin E (38), garlic (39), red wine, and purple grape juice(40). Herbal and dietary supplements may both interfere with or enhancethe effect of aspirin on platelets.

Janssen, et al provided healthy volunteers with 3 mg of aspirin per day,a study designed to mimic acetylsalicylic acid levels in certain plants.(41) They showed that serum TXB₂ levels were reduced by 39% compared toplacebo.

A number of prospective randomized clinical trials have demonstratedthat 50 to 500 milligrams of aspirin per day effectively reduces therisk of primary or secondary arterial thrombotic events in manyindividuals. Recognition of aspirin's antithrombotic properties hasprompted clinical researchers to focus substantial efforts towardsmodifying platelet function with not only aspirin but a variety of newlydeveloped platelet-suppressive drugs.

Despite the availability of these new drugs, the use of aspirin forarterial thrombosis prevention continues to increase as public awarenessgrows. More than 80 billion aspirin tablets are consumed annually in theUSA, and more than 37% of the individuals taking aspirin do so to“prevent blood clots”. Additionally, individuals who employ alternativemedicine practices may consume significant quantities of red wine,purple grape juice, fish oil, vitamin E, garlic, ginkgo biloba, andother substances known to interfere with platelet function.

Numerous reports in the scientific literature detail varied individualresponses to aspirin dosages. However, little is reported regarding thepotential need to adjust aspirin dosage according to individual biologicresponse or to a changing response over time. Limited studies correlatethe occurrence of thrombotic events with individuals who become“resistant” to aspirin.

Subject 3 8 12 14 17 18 24 % Inhibition (81) 60 78 46 48 70 17 10 %Inhibition (325) 44 40 82 83 46 83 38

Why hasn't aspirin, the drug most widely consumed to modify plateletfunction, been dosed according to biologic response, in a manner similarto anticoagulants? Because most laboratory tests used to monitorplatelet function are time consuming, require expensive equipment(platelet aggregometer), or are traumatic to the patient (bleedingtime). In addition, all current assay methods are subject to widevariation in results due to preanalytical and analytical variables,inherent in all ex vivo tests that require the patient's platelets.

SUMMARY OF THE INVENTION

The present invention, in a general and overall sense, provides a methodfor identifying an optimal minimal aspirin dose for a patient that isspecifically tailored to the patient's specific platelet response levels(i.e., to monitor platelet in activation).

The method includes measurement of thromboxane B₂ metabolite levels inthe patient to determine the optimum dose for platelet inhibition, withminimum aspirin related side-effects. It is envisioned that theinvention, in one aspect, may also be provided in a test kit form. Insome embodiments, the kit will be provided together with a solidsubstrate that is at least partially coated with a material such as anantibody, that is capable of reacting with a thromboxane B₂ metabolite,such as 11-dehydro TXB₂. Also included would be a reactor fluid, such asone that would change in color upon exposure to the thromboxane B₂metabolite.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Biochemical pathway in a cell showing release of 11-dehydrothromboxane B₂ from thromboxane B₂ in the presence of dehydrogenase

FIG. 2—Biochemical pathway showing the reaction products, salicylicacidand acetylated cycloxygenase from cycloxygenase and acetylsalicylic acid

FIG. 3—pg 11-dehydro TXB₂/mg creatinine ((x axis) vs frequency (y axis)

FIG. 4—□=Baseline

-   -   ▮=Post 81 mg

FIG. 5—□=Baseline

-   -   ▮=Post 325 mg

FIG. 6—□=Baseline

-   -   ▮=Post 81 mg

(Shaded Box) □=Post 325 mg

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Measuring Platelet Activation Platelet Aggregometry and invitro Activation

One may detect and measure in vitro platelet activation usingaggregometry, which detects platelet aggregation; lumiaggregometry,which detects both aggregation and platelet-specific secretions; orplatelet-activation instrumentation such as the Dade-Behring PFA-100platelet function analyzer.

Platelet Activation Aetabolites and in vitro Activation

Plasma, serum, or urine assays of platelet secretions and arachidonicacid pathway metabolites may be employed in the detection of in vitroplatelet activation. Assays of plasma or serum TXB₂ are used extensivelyin platelet function research laboratories where specimen management iscarefully controlled, but routine clinical measurement is hampered bythe in vitro instability of platelets.

Aspirin Dosage and Measures of Platelet Activity

Measurement of aspirin-induced platelet suppression has led to someunexpected findings. Using the arachidonic acid metabolite 12-L-5, 8,10-heptadecatrienoic acid (12-HHT) as marker, Beving et. al. measuredaspirin's suppression of platelet activity at three dosages, 30, 75, and150 mg/day. (42) His group demonstrated that, after seven days'treatment, discontinuing the higher dosages triggered a reboundphenomenon. This was reflected in significantly elevated 12-HHT levelspersisting for up to six weeks after stopping aspirin therapy. Further,they demonstrated the rebound to be greater in patients whose baselinelevels of 12-HHT were elevated. They concluded that the degree ofplatelet suppression and ultimate rebound effect could be controlled bydetermining the pre-aspirin platelet activity via 12-HHT level analysisand by adjusting aspirin dosage accordingly. The rebound effect did notoccur in the 30 mg/day dosage arm.

Buerke et. al. used bleeding times, platelet aggregometry, and serumTXB₂ assays to demonstrate effective aspirin dosages in healthy males.(43) Comparing various dosage combinations, they recommended a loadingdose of 300 mg aspirin in combination with 40 mg/day as a maintenancedose to achieve optimum suppression of platelets. Loading dosages of 40or 100 mg failed to elicit significant changes within two hours ofadministration.

The Urinary Activation Marker 11-dehydro Thromboxane B₂

Whole blood or plasma specimens for platelet metabolite assays requirespecial management because the platelets tend to become activated bychanges in temperature, exposure to non-biological surfaces, and mildmechanical agitation. Aggregometry assays are qualitative andtechnology-intensive, and bleeding time tests have poor predictivevalues. Thus, it is necessary to locate a metabolite formed in vivo fromthe products of platelet activation. (45)

Hepatocyte 11-hydroxy thromboxane dehydrogenase acts upon plasma TXB₂ toproduce 11-dehydro TXB₂. (46) The plasma half-life of 11-dehydro TXB₂ is45 minutes, and plasma levels remain in the nanogram range, as it israpidly cleared by the kidney. (47) The urine concentration of 11-dehydro TXB₂, however, is plentiful and, as platelets appear to be itsonly source, proportionally reflects platelet activity within theprevious twelve hours. (46) Urine levels of 11 -dehydro TXB₂ arefrequently elevated in atherosclerosis, the chronic phase followingstroke, transient ischemic attack, intracerebral hemorrhage, and atrialfibrillation. (48) Further, 11-dehydro TXB₂ levels are typicallydecreased in aspirin therapy, even in cases of atherosclerosis,myocardial infarction, and atrial fibrillation.

Assays for 11-dehydro TXB₂ have intra- and interassay CVs of ≦10% and donot cross-react with TXB₂, 2, 3-dinor TXB₂, nor other cyclooxygenasemetabolites. (49).

EXAMPLE 2 11-dehydro TXB₂ in Randomly Collected Urine from Aspirin andNon-Aspirin Donors

Urine 11-dehydro TXB₂ was assayed in random urine specimens to:

-   -   Establish a reference range in non-aspirin users.    -   Determine whether aspirin therapy is related to levels below the        reference range.    -   Detect aspirin resistance in individuals taking aspirin therapy.        Materials and Methods        11-dehydro TXB₂ Reference Range and Results in Aspirin Treatment

Random urine specimens were collected from 65 individuals who hadavoided aspirin for at least two weeks and from 45 individuals who weretaking 81 or 325 mg/day by prescription. Each specimen was assayed for11-dehydro-TXB₂. Specimens were assayed at two dilutions usingacetylcholinesterase-linked enzyme immunoassay; the results were testedfor parallelism and averaged. Urine creatinine was assayed using theJaffe picrate reaction. To normalize for urinary output, results wereexpressed in pg 11-dehydro TXB₂/mg creatinine. Results of allpopulations were compared using the student t-test. Within-day variationis 30% and day to day variation 20%.

Effect of Aspirin on 11-dehydro TXB2 in Healthy Non-aspirin Users

In a crossover study, twenty-four healthy individuals who had avoidedaspirin for a minimum of ten days collected random baseline urinespecimens. They then took one 81 or 325 mg tablet at 8:00 AM andcollected a second urine specimen 24 hours after the aspirin was taken.Following a 2-week washout period dosages were reversed and anotherbaseline and 24-hours post-aspirin specimen was collected.

Results

Reference Range and Aspirin Values

Individuals taking 81 to 325 mg/day aspirin exhibit significantly lowerlevels of 11-dehydro-TXB₂ than those not taking aspirin. See Table 1.

TABLE 1 Aspirin therapy Aspirin non-users p-value N  45  65 Mean 5232080 <0.00001 SD 514 1360

A decision point of 1000 pg 11-dehydro-TXB₂/mg creatinine yields bothfalse negative and false positive rates of 10%, the best achievablecombination, as shown in Table 2. Aspirin effect is ruled in at adecision point of 800 pg/mg or less and ruled out of 1000 pg/mg. FIG. 3is a histogram that illustrates the clear separation between thenon-aspirin and aspirin population. Of the aspirin users, sixindividuals (13%) bad results above the decision point and two exceededthe aspirin effect rule-out point of 1000. These six may beaspirin-resistant, and are being observed closely for potentialthrombotic events.

TABLE 2 False True True False Positive Positive Positive NegativeDecision rate for Rate for Rate for rate for level in aspirin Aspirinaspirin aspirin pg/mg effect Effect effect effect Aspirin rule-in 6000.0% 75.6% 100.0% 24.4% ≦800 pg/mg 700 0.0% 80.5% 100.0% 19.5% 800 0.0%80.5% 100.0% 19.5% 900 1.8% 85.4% 98.2% 14.6% Recommended 1000 10.5%90.2% 89.5% 9.8% Decision point 1100 17.5% 90.2% 82.5% 9.8% 1200 24.6%95.1% 75.4% 4.9% 1300 29.8% 97.6% 70.2% 4.9% 1400 33.3% 97.6% 66.7% 2.4%Aspirin rule-out 1500 35.1% 100.0% 64.9% 0.0% ≧1500 pg/mg 1600 42.1%100.0% 57.9% 0.0%

EXAMPLE 3 Effect of Aspirin on the 11 Dehydro TXB₂ Level of HealthyNon-aspirin Users

Initiation of aspirin therapy causes mean reduction of 68% for 81 mg/dayand 76% for 325 mg/day as illustrated in FIGS. 4 and 5. There was nosignificant difference between the 81 mg/day and 325 mg/day suppressionlevels.

An immunoassay has been investigated here that measures the effect ofaspirin on platelet function. A stable metabolite of the plateletactivation process, 11-dehydro-TXB₂, can be measured in random urine,bypassing the need for ex vivo platelet function. Utilizing this assaywe have been able to show a significant difference between individualstaking 81 or 325 mg of aspirin and individuals not taking aspirin.Values equal to or less than 800 pg 11-dehydro-TXB₂/mg creatinineindicates that aspirin usage is sufficiently inhibiting COX-1 activity.Aspirin users with levels over 1000 pg/mg appear to be resistant and notachieving optimal platelet inhibition, as demonstrated in FIG. 3.Utilizing these decision point criteria, we define 13% of individuals aspotential non-responders, coinciding with other published reports.

The present findings demonstrate that the range of non-aspirin resultsis broad and appears bimodally distributed. The higher peak representsincreased platelet activation.

From the crossover study, seven individuals (30%) had a less than 50%response to either 81 mg or 325 mg of aspirin. Of these, threedemonstrated less 11-dehydro TXB₂ excretion at 81 mg than at 325 mg.However, one individual (subject 24) did not demonstrate 50% reductionto 81 mg or 325 mg of aspirin suggesting aspirin resistance. Theinhibitory contribution of dietary and lifestyle habits should always betaken into consideration when interpreting data and may have contributedto these results. Further, urine metabolite interference, exercise, andhormonal variation may be responsible for a 20-30% within-day andday-to-day variation

Aspirin antithrombotic therapy is long-term therapy. The urine11-dehydro TXB₂ essay is a readily available means for evaluatingindividual response to aspirin. It is useful for detecting aspirinresistance and for prescribing and monitoring alternate therapies. Theassay is also useful in monitoring patient compliance and determiningthe lowest aspirin dose that produces effective COX-1 inhibition whileavoiding aspirin's unpleasant, sometimes dangerous side effects.

Using the 11-dehydro TXB₂ assay as the measure of platelet activity, werecommend clinical trials that 1) further compare biologic response toaspirin's affect on clinical outcomes, 2) evaluate whether dosageadjustment according to COX-1 inhibition will reduce thrombotic eventswhile limiting untoward side effects, and 3) determine whether theinfluence of inflammatory process, reflected by abnormal C-reactiveprotein levels, are associated with aspirin resistance.

Aspirin has been proven to prevent secondary arterial thrombosis in avariety of conditions, and may be used as defined here to preventprimary heart attacks and strokes to those who are at risk. Propermonitoring of aspirin therapy will lead to more accurate dosing,prevention of side effects, and the management of aspirin resistance.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents, which are both chemically and physiologically, relatedmay be substituted for the agents described herein while the same orsimilar results would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

EXAMPLE 4

The concept of utilizing 11-dehydrothromboxane measurements to determineindividuals in whom aspirin or other platelet modifying compounds is nothaving a significant platelet inhibiting effect may have severaladditional applications.

The antigen antibody assay currently performed by conventional ELISAtechnique could also be performed using a point of care instrument. Thetest method would have to be modified such that the reaction could takeplace and the end point be determined within the confines of the pointof care instrument.

Additionally, it may be possible to adapt the assay for patient selftesting utilizing “dip stick” technology similar to that used forglucose.

EXAMPLE 5 Urinary Thromboxane B₂ Metabolite Monitors the Anti-plateletAction of Aspirin

Concern for side effects and laboratory evidence for aspirin“resistance” in up to 20% of individuals make it essential to establisha simple, effective laboratory monitoring system for aspirin'santithrombotic efficacy. The present example demonstrates the utility ofthe invention for the purpose in human patients. 11-dehydrothromboxaneB₂, (11-de-H-TXB₂), a stable metabolite of platelet activation wasmeasured in random urine specimens from 65 people who were not takingaspirin and 45 who were taking at least 81 mg/day. The means fornon-aspirin users was 2080 pg 11-de-H-TXB₂/mg creatinine, and foraspirin users was 523, p.<0.0000.1. Receiver-operating characteristicanalysis yields an optimum decision point of 1000 pg 11-de-H-TXB₂ inrandom urine may be used to document aspirin's platelet-suppressiveeffects and to identify individuals who may not achieve antithromboticbenefits.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for determining a minimum optimum aspirin dose forinhibiting platelet activation in a patient, said method comprising: a)administering to a patient a known dose of aspirin; b) collecting aurine sample from the patient; c) determining an amount of a thromboxaneB₂ metabolite in the urine sample; d) determining an amount ofcreatinine in the urine sample; e) normalizing the amount of thromboxaneB₂ metabolite to the amount of creatinine to create a thromboxane B₂metabolite/creatinine ratio; f) optionally repeating steps a) through e)with different known doses of aspirin to determine the optimum dose ofaspirin which is the dose that provides less than about 1000 pgthromboxane B₂ metabolite/mg creatinine.
 2. The method of claim 1,wherein the thromboxane B₂ metabolite is 11-dehydrothromboxane B₂. 3.The method of claim 1, wherein the patient is a post-thrombotic eventpatient.
 4. The method of claim 1, wherein the patient has had ischemia,intracerebral hemorrhage, arterial fibrillation, cardiaccatheterization, lupus, or coronary angioplasty.
 5. The method of claim1, comprising determining the optimum dose of aspirin which is the dosethat provides less than about 800 pg thromboxane B₂ metabolite/mgcreatinine.
 6. The method of claim 1, comprising determining the optimumdose of aspirin which is the dose that provides less than about 500 pgthromboxane B₂ metabolite/mg creatinine.
 7. The method of claim 1,comprising determining the optimum dose of aspirin which is the dosethat provides less than about 400 pg thromboxane B₂ metabolite/mgcreatinine.
 8. The method of claim 1, wherein the known dose of aspirinis between about 80 mg and about 325 mg.
 9. The method of claim 1,wherein the known dose of aspirin is between 81 mg or about 325 mg. 10.The method of claim 1, wherein the known dose of aspirin is about 80 mg,about 200 mg, or about 300 mg.